Biomedical subjects

Jeremy S Myers

Publications and source records attributed to Jeremy S Myers.

8 recordsLinked to original sources

PubMed2006

Rapid activation of ATR by ionizing radiation requires ATM and Mre11.

The ataxia-telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR) protein kinases are crucial regulatory proteins in genotoxic stress response pathways that pause the cell cycle to permit DNA repair. Here we show that Chk1 phosphorylation in response to hydroxyurea and ultraviolet radiation is ATR-dependent and ATM- and Mre11-independent. In contrast, Chk1 phosphorylation in response to ionizing radiation (IR) is dependent on ATR, ATM, and Mre11. The ATR and ATM/Mre11 pathways are generally thought to be separate with ATM activation occurring early and ATR activation occurring as a late response to double strand breaks. However, we demonstrate that ATR is activated rapidly by IR, and ATM and Mre11 enhance ATR signaling. ATR-ATR-interacting protein recruitment to double strand breaks is less efficient in the absence of ATM and Mre11. Furthermore, IR-induced replication protein A foci formation is defective in ATM- and Mre11-deficient cells. Thus, ATM and Mre11 may stimulate the ATR signaling pathway by converting DNA damage generated by IR into structures that recruit and activate ATR.

Antineoplastic Agents↗

PubMed2005

ATRIP binding to replication protein A-single-stranded DNA promotes ATR-ATRIP localization but is dispensable for Chk1 phosphorylation.

ATR associates with the regulatory protein ATRIP that has been proposed to localize ATR to sites of DNA damage through an interaction with single-stranded DNA (ssDNA) coated with replication protein A (RPA). We tested this hypothesis and found that ATRIP is required for ATR accumulation at intranuclear foci induced by DNA damage. A domain at the N terminus of ATRIP is necessary and sufficient for interaction with RPA-ssDNA. Deletion of the ssDNA-RPA interaction domain of ATRIP greatly diminished accumulation of ATRIP into foci. However, the ATRIP-RPA-ssDNA interaction is not sufficient for ATRIP recognition of DNA damage. A splice variant of ATRIP that cannot bind to ATR revealed that ATR association is also essential for proper ATRIP localization. Furthermore, the ATRIP-RPA-ssDNA interaction is not absolutely essential for ATR activation because ATR phosphorylates Chk1 in cells expressing only a mutant of ATRIP that does not bind to RPA-ssDNA. These data suggest that binding to RPA-ssDNA is not the essential function of ATRIP in ATR-dependent checkpoint signaling and ATR has an important function in properly localizing the ATR-ATRIP complex.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Straightening out the LINEs: LINE-1 orthologous loci.

The L1Hs preTa subfamily of long interspersed elements (LINEs) originated after the divergence of human and chimpanzee and is therefore found only in the human genome. Thirty-three of the 254 L1Hs preTa elements are polymorphic for the absence/presence of the insertion, making them useful markers for studying human population genetics. The problem of homoplasy, however, can diminish the value of LINEs as phylogenetic and population genetic markers. We examined anomalous orthologous sites in a range of nonhuman primates. Only two cases of other mobile elements inserting near the preintegration sites of L1Hs preTa elements were observed: an AluY insertion in Chlorocebus and an L1PA8 insertion in Aotus. Sequence analysis showed that both elements were clearly distinguishable from their human counterparts. We conclude that L1 elements can continue to be regarded as essentially homoplasy-free genetic characters.

Animals↗

PubMed2003

Alu elements and hominid phylogenetics.

Alu elements have inserted in primate genomes throughout the evolution of the order. One particular Alu lineage (Ye) began amplifying relatively early in hominid evolution and continued propagating at a low level as many of its members are found in a variety of hominid genomes. This study represents the first conclusive application of short interspersed elements, which are considered nearly homoplasy-free, to elucidate the phylogeny of hominids. Phylogenetic analysis of Alu Ye5 elements and elements from several other subfamilies reveals high levels of support for monophyly of Hominidae, tribe Hominini and subtribe Hominina. Here we present the strongest evidence reported to date for a sister relationship between humans and chimpanzees while clearly distinguishing the chimpanzee and human lineages.

Alu Elements↗

PubMed2003

Following the LINEs: an analysis of primate genomic variation at human-specific LINE-1 insertion sites.

The L1 Ta subfamily of long interspersed elements (LINEs) consists exclusively of human-specific L1 elements. Polymerase chain reaction-based screening in nonhuman primate genomes of the orthologous sites for 249 human L1 Ta elements resulted in the recovery of various types of sequence variants for approximately 12% of these loci. Sequence analysis was employed to capture the nature of the observed variation and to determine the levels of gene conversion and insertion site homoplasy associated with LINE elements. Half of the orthologous loci differed from the predicted sizes due to localized sequence variants that occurred as a result of common mutational processes in ancestral sequences, often including regions containing simple sequence repeats. Additional sequence variation included genomic deletions that occurred upon L1 insertion, as well as successive mobile element insertions that accumulated within a single locus over evolutionary time. Parallel independent mobile element insertions at orthologous loci in distinct species may introduce homoplasy into retroelement-based phylogenetic and population genetic data. We estimate the overall frequency of parallel independent insertion events at L1 insertion sites in seven different primate species to be very low (0.52%). In addition, no cases of insertion site homoplasy involved the integration of a second L1 element at any of the loci, but rather largely involved secondary insertions of Alu elements. No independent mobile element insertion events were found at orthologous loci in the human and chimpanzee genomes. Therefore, L1 insertion polymorphisms appear to be essentially homoplasy free characters well suited for the study of population genetics and phylogenetic relationships within closely related species.

Animals↗

PubMed2003

LINE-1 preTa elements in the human genome.

The preTa subfamily of long interspersed elements (LINEs) is characterized by a three base-pair "ACG" sequence in the 3' untranslated region, contains approximately 400 members in the human genome, and has low level of nucleotide divergence with an estimated average age of 2.34 million years old suggesting that expansion of the L1 preTa subfamily occurred just after the divergence of humans and African apes. We have identified 362 preTa L1 elements from the draft human genomic sequence, investigated the genomic characteristics of preTa L1 insertions, and screened individual elements across diverse human populations and various non-human primate species using polymerase chain reaction (PCR) assays to determine the phylogenetic origin and levels of human genomic diversity associated with the L1 elements. All of the preTa L1 elements analyzed by PCR were absent from the orthologous positions in non-human primate genomes with 33 (14%) of the L1 elements being polymorphic with respect to insertion presence or absence in the human genome. The newly identified L1 insertion polymorphisms will prove useful as identical by descent genetic markers for the study of human population genetics. We provide evidence that preTa L1 elements show an integration site preference for genomic regions with low GC content. Computational analysis of the preTa L1 elements revealed that 29% of the elements amenable to complete sequence analysis have apparently escaped 5' truncation and are essentially full-length (approximately 6kb). In all, 29 have two intact open reading frames and may be capable of retrotransposition.

Animals↗

PubMed2002

A comprehensive analysis of recently integrated human Ta L1 elements.

The Ta (transcribed, subset a) subfamily of L1 LINEs (long interspersed elements) is characterized by a 3-bp ACA sequence in the 3' untranslated region and contains approximately 520 members in the human genome. Here, we have extracted 468 Ta L1Hs (L1 human specific) elements from the draft human genomic sequence and screened individual elements using polymerase-chain-reaction (PCR) assays to determine their phylogenetic origin and levels of human genomic diversity. One hundred twenty-four of the elements amenable to complete sequence analysis were full length ( approximately 6 kb) and have apparently escaped any 5' truncation. Forty-four of these full-length elements have two intact open reading frames and may be capable of retrotransposition. Sequence analysis of the Ta L1 elements showed a low level of nucleotide divergence with an estimated age of 1.99 million years, suggesting that expansion of the L1 Ta subfamily occurred after the divergence of humans and African apes. A total of 262 Ta L1 elements were screened with PCR-based assays to determine their phylogenetic origin and the level of human genomic variation associated with each element. All of the Ta L1 elements analyzed by PCR were absent from the orthologous positions in nonhuman primate genomes, except for a single element (L1HS72) that was also present in the common (Pan troglodytes) and pygmy (P. paniscus) chimpanzee genomes. Sequence analysis revealed that this single exception is the product of a gene conversion event involving an older preexisting L1 element. One hundred fifteen (45%) of the Ta L1 elements were polymorphic with respect to insertion presence or absence and will serve as identical-by-descent markers for the study of human evolution.

Animals↗

PubMed2002

DNA repair mediated by endonuclease-independent LINE-1 retrotransposition.

Long interspersed elements (LINE-1s) are abundant retrotransposons in mammalian genomes that probably retrotranspose by target site-primed reverse transcription (TPRT). During TPRT, the LINE-1 endonuclease cleaves genomic DNA, freeing a 3' hydroxyl that serves as a primer for reverse transcription of LINE-1 RNA by LINE-1 reverse transcriptase. The nascent LINE-1 cDNA joins to genomic DNA, generating LINE-1 structural hallmarks such as frequent 5' truncations, a 3' poly(A)+ tail and variable-length target site duplications (TSDs). Here we describe a pathway for LINE-1 retrotransposition in Chinese hamster ovary (CHO) cells that acts independently of endonuclease but is dependent upon reverse transcriptase. We show that endonuclease-independent LINE-1 retrotransposition occurs at near-wildtype levels in two mutant cell lines that are deficient in nonhomologous end-joining (NHEJ). Analysis of the pre- and post-integration sites revealed that endonuclease-independent retrotransposition results in unusual structures because the LINE-1s integrate at atypical target sequences, are truncated predominantly at their 3' ends and lack TSDs. Moreover, two of nine endonuclease-independent retrotranspositions contained cDNA fragments at their 3' ends that are probably derived from the reverse transcription of endogenous mRNA. Thus, our results suggest that LINE-1s can integrate into DNA lesions, resulting in retrotransposon-mediated DNA repair in mammalian cells.

Animals↗